SPECIFIC REDUCTION OF CHLOROPLAST GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE-ACTIVITY BY ANTISENSE RNA REDUCES CO2 ASSIMILATION VIA A REDUCTION IN RIBULOSE-BISPHOSPHATE REGENERATION IN TRANSGENIC TOBACCO PLANTS

The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylak oid membranes with the carbon metabolism of the photosynthetic carbon- reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydr ogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enz ymes which catalyse this reversible conversion. We report on the engin eering of an antisense RNA construct directed against the tobacco (Nic otiana tabacum L.) chloroplast-located GAPDH (A subunit). The construc t was integrated into the tobacco genome by Agrobacterium-mediated tra nsformation of leaf discs. Of the resulting transfermants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% o f wild-type (WT) activities. Segregation analysis of the kanamycin-res istance character in self-pollinated T-1 seed from each of the five tr ansformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T-1 progeny from GAP-R was used to gen erate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH a ctivities on metabolite pools for ribulose-1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO2 assimilation and other gas-exchange parameters. The RuBP pool size was linearly relate d to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO2 assimilation was not affected until RuBP levels dropped to 30-40% of WT levels. That is, the CO2 assimilation r ate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol(mol site)(-1) while the ratio for WT p lants was 4- 5 mol.m(mol site)(-1). Leaf conductance was not reduced i n leaves with reduced GAPDH activities, resulting in an increase in th e ratio of intercellular to ambient CO2 partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO2 and showed a normal decline with increases in CO2 partial pressure. Altho ugh PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas- exchange and chlorophyll a fluorescence measurements indicated that ph otosynthesis changed from being Rubisco-limited to being RuBP-regenera tion-limited at a lower CO2 partial pressure in the antisense plants t han in WT plants. Photosynthetic electron transport was down-regulated by the build-up of a large proton gradient and the electron-transport chain did not become over-reduced due to a shortage of NADP. Plants w ith severely reduced GAPDH activity were not photoinhibited despite th e continuous presence of a large thylakoid proton gradient in the ligh t. Along with plant size, Rubisco activity, leaf soluble protein and c hlorophyll content were reduced in plants with the lowest GAPDH activi ties. We conclude that chloroplastic GAPDH activity does not appear to limit steady-state photosynthetic CO2 assimilation at ambient CO2. Th is is because WT leaves maintain the ratio of RuBP per Rubisco site ab out twofold higher than the level required to achieve a maximal rate o f CO2 assimilation.